This invention relates to peptide synthesis, in particular to the synthesis of a peptide hormone. The invention relates especially to the synthesis of a peptide of the insulin family, particularly to the synthesis of relaxin.
Relaxin (RLX) was discovered in 1926 by Frederick Hisaw [Hisaw, F. (1926) Experimental relaxation of the pubic ligament of the guinea pig. Proc. Soc. Exp. Biol. Med. 23, 661-663] as a substance that could relax the pelvic ligaments and regulate the female reproductive tract functions. The relaxin family of peptides comprises the relaxin-1 (RLX1), relaxin-2 (RLX2) and relaxin-3 (RLX3). Relaxin peptides belong to the greater family of the insulin like peptides (INSL). This peptide family includes insulin and insulin like peptide 3, 4, 5 and 6. These peptides have a high degree of structural similarity.
In addition to the female reproductive tract function, relaxins are known to participate in a range of medical conditions for example in cardiac protection, as disclosed in Samuel, C. S. and Hewitson, T. D. (2006) Relaxin in cardiovascular and renal disease; Kidney Int. 69, 1498-1502; Bani, D., Nistri, S., Bani Sacchi, T. and Bigazzi, M. (2005) Basic progress and future therapeutic perspectives of relaxin in ischemic heart disease. Ann. N. Y. Acad. Sci. 1041, 423-430; Samuel, C. S., Du, X. J., Bathgate, R. A. D. and Summers, R. J. (2006) “Relaxin” the stiffened heart and arteries: the therapeutic potential for relaxin in the treatment of cardiovascular disease. Pharmacol. Ther. 112, 529-552; Dschietzig, T., Bartsch, C., Baumann, G. and Stangl, K. (2006) Relaxin—a pleiotropic hormone and its emerging role for experimental and clinical therapeutics. Pharmacol. Ther. 112, 38-56; in fibrosis as disclosed in Bathgate, R. A. D., Hsueh, A. J. and Sherwood, O. D. (2006) Physiology and molecular biology of the relaxin peptide family. In: Physiology of Reproduction. (Knobil, E. and Neill, J. D., Eds), 679-770. Elsevier, San Diego; Sherwood, O. D. (2004) Relaxins physiological roles and other diverse actions. Endocr. Rev. 25, 205-234; Samuel, C. S. (2005) Relaxin: antifibrotic properties and effects in models of disease. Clin. Med. Res. 3, 241-249; in allergic responses as disclosed in Bani, D. (1997) Relaxin: a pleiotropic hormone. Gen. Pharmacol. 28, 13-22.; in cancer as disclosed in Silvertown, J. D., Summerlee, A. J. and Klonisch, T. (2003) Relaxin-like peptides in cancer. Int. J. Cancer 107, 513-519; Kamat, A. A., Feng, S., Agoulnik, I. U., Kheradmand, F., Bogatcheva, N. V., Coffey, D., Sood, A. K. and Agoulnik, A. I. (2006) The role of relaxin in endometrial cancer. Cancer Biol. Ther. 5, 71-77; and in wound healing as disclosed in Yamaguchi, Y. and Yoshikawa, K. (2001) Cutaneous wound healing: an update. J. Dermatol. 28, 521-534; 113 Wyatt, T. A., Sisson, J. H., Forget, M. A., Bennett, R. G., Hamel, F. G. and Spurzem, J. R. (2002) Relaxin stimulates bronchial epithelial cell PKA activation, migration, and ciliary beating, Exp. Biol. Med. (Maywood) 227, 1047-1053; Casten, G. G. and Boucek, R. J. (1958) Use of relaxin in the treatment of scleroderma. J. Am. Med. Assoc. 166, 319-324.
Other therapeutic applications of RLX2 are believed to be associated with its ability to control collagen turnover as disclosed in Samuel C S, Hewitson T D, Unemori E N, Tang M L, Cell Mol Life Sci. 2007, 64, 1539-57. Drugs of the future: the hormone relaxin.
RLX2 potentially has a wide range of therapeutic applications and a significant demand exists for its use in research and for therapeutic purposes. The therapeutic potential of other relaxins has generally not been investigated due to difficulties in producing or isolating them.
RLX has two peptide chains, generally referred to as the A chain (RLXA) and the B chain (RLXB). The chains are joined by two intermolecular cysteine bridges and chain A contains an additional intramolecular disulphide bond. The conformational arrangement of the chains is an important feature of relaxins particularly RLX1 and RLX2 and the two chains must be connected with the appropriate disulphide bonds in order to exhibit the appropriate biological activity. Furthermore RLXB is generally highly insoluble in aqueous solution. The insolubility of RLXB and the need to ensure the appropriate disulphide bonds are formed means synthesis by random chain combination is very difficult and makes the purification of RLXB, for example by chromatographic methods, very difficult, as disclosed in J.-G. Tang et al, Biochemistry 2003, 42, 2731-2739; Wade, J. D., and Tregear, G. W. (1997) Relaxin. Methods Enzymol. 289, 637-646.
Methods of production of relaxins using recombinant DNA techniques have been disclosed in U.S. Pat. Nos. 4,758,516 and 5,023,321 a division of U.S. Pat. No. 4,758,516. In these patents, genes and DNA transfer vectors for the expression of human preprorelaxin and subunits thereof including genes and transfer vectors for the expression of human prorelaxin and the A, B and C chains are disclosed with methods for synthesis of the peptides using recombinant DNA techniques.
U.S. Pat. No. 5,464,756 discloses a process for cleaving a peptide into two polypeptide components by treating a reduced free-cysteine form of the polypeptide with a cleaving agent and in particular culturing cells containing DNA encoding the polypeptide and having at least one Asp codon present at the position to be cleaved such that DNA is expressed to produce the polypeptide in the host cell culture and treating the free-cysteine form of the polypeptide with dilute acid to effect the desired cleavage.
Recombinant DNA techniques may be lengthy and complex and unsatisfactory for production of relaxins on a large scale. Furthermore, as materials used in the techniques are animal-based, objections to the use of relaxins produced by such methods may arise on religious grounds or for ethical reasons, limiting the utility of relaxin products produced in this way.
Chemical synthesis of relaxins generally has proved problematic. Chemical synthesis of RLX1 is not known and consequently nor is the investigation of possible therapeutic uses of synthetic RLX1.
E. Bullesbach and C. Schwabe, Journal Biol. Chem. 1991, 266, 10754-10761; E. Bullesbach and C. Schwabe, J. Biol. Chem. 2005, 280, 14586-14590 discloses the chemical synthesis of RLX 2. This process involves the solid phase synthesis of the two individual chains and their site directed combination that is protecting a specific cysteine residue to ensure pre-determined cysteine residues combine to form a specific disulphide link. After the assembly of the chains, two reaction steps requiring the application of hydrogen fluoride and three reaction steps for the site directed chain combination are needed for the completion of the synthesis of RLX2. This method is however, very laborious, has poor yields and undesirably requires the use of highly toxic and hazardous hydrogen fluoride.
U.S. Pat. No. 4,835,251 discloses a method for combining an A chain of human relaxin and a B chain of human relaxin to produce biologically active human relaxin by mixing a reduced free-cysteine form of the A chain and a reduced free-cysteine form of the B chain in an aqueous medium at a pH of 7 to 12 under oxygen wherein the B chain, but not the product, is denatured.
Attempts to produce synthetic human relaxins have however not yielded satisfactory results. Chain B of human relaxin-1 (RLX1B) and of human relaxin-2 (RLX2B) and intermediate smaller peptides and fragments are highly insoluble or hydrophobic and difficulties have been encountered in extending the peptide chain around the sequence Ala-Gln-Ile-Ala-Ile-Cys (SEQ ID NO:1) of RLX1B and RLX2B. Solid phase synthesis routes involve very difficult coupling and deprotection steps. Furthermore, difficulties are encountered in forming the appropriate interchain disulphide Bond combinations for RLX1B and RLX2B with the corresponding relaxin A-chains due to the insolubility of the B chains leading to undesirable precipitation or non-dissolution of B chains during synthesis of the relaxin.